Plant and Cell Physiology

Endogenous circadian rhythms in plants play a role in adaptation to day-night cycles. The circadian clock is a cell-autonomous system that functions through the coordination of time information in the plant body. Synchronization of cellular clocks is based on coordination mechanisms; the synchronization manners in proliferating plants remain unclear. We performed spatiotemporal analysis of the circadian rhythm of fronds (leaf-like plant units) of proliferating Lemna minor plants carrying a circadian bioluminescence reporter, AtCCA1:LUC. Noninvasive observations of the bioluminescence of fast-growing two-dimensional plants allowed us to analyze the circadian rhythms at a cell-level resolution and obtain information regarding frond lineage. We focused on spontaneous circadian organization under constant light conditions for plants with light/dark treatment (LD-grown) or without it (LL-grown). Even fronds developing from an LL-grown parental frond showed coherent circadian rhythms among them. This allowed the maintenance of circadian rhythmicity in proliferating plants. Inside a frond, a centrifugal phase/period pattern was observed in LD-grown plants, whereas various phase patterns with traveling waves were formed in LL-grown plants. These patterns were model-simulated by local coupling of cellular circadian oscillators with different initial synchronous states in fronds. Taken together with similar patterning previously reported for detached leaves of Arabidopsis, it is strongly suggested that local coupling is the primary force for the development of these phase patterns in plants lacking long-distance communication. We propose a basic framework of spontaneous phase patterning with three stages of circadian organization: initial phasing, evolution of patterning, and desynchronization/randomizing of phase, in association with altering cell-cell coupling.

Iron-sulfur (Fe-S) clusters are essential cofactors for Fe-S proteins. SUFBC2D complex is the scaffold responsible for Fe-S cluster assembly in chloroplasts. However, the regulatory mechanism on SUFBC2D remains elusive. In this study, we report that the transcription of SUFB responds rapidly to leaf senescence, whereas the transcription of SUFC and SUFD does not. Intriguingly, their protein contents remain stable during leaf senescence. We further found that leaf death was occurred only when SUFB RNAi was induced, and SUFB and SUFC contents decreased much faster in the SUFB-RNAi lines than in the SUFC-RNAi lines, indicating that SUFB had a faster turnover rate than SUFC. Moreover, overexpressing SUFB increased the contents of SUFC and SUFD, and SUFBC2D, whereas overexpressing SUFC did not increase SUFB and SUFD. Our findings reveal that SUFB stabilizes SUFC and SUFD via forming SUFBC2D, whereas SUFC lacks this function. Furthermore, SUFB expression was sharply downregulated when the plants were subjected to iron deficiency, whereas SUFC and SUFD expression was not. Interestingly, the contents of all three SUF members decreased, indicating that plants degrade SUFBC2D in response to iron deficiency by downregulating SUFB transcription. We subsequently studied the degradation mechanism of SUFBC2D. Our results indicated that all SUFs are substrates of the caseinolytic protease (CLP) because they all accumulated in the CLP impaired mutant, and they physically interact with CLPS1, the substrate recognition adaptor of CLP. Collectively, our findings provide novel insights into how plants regulate SUFBC2D complex via SUFB to adapt to leaf senescence and iron deficiency.

The circadian clock regulates temporal metabolic activities, enabling organisms to adapt to cyclic environmental changes, but how it affects lipid metabolism in plants is poorly understood. Our previous finding showed that the central clock transcription factors LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) increased seed oil contents in Arabidopsis. Here we investigated the molecular and metabolic mechanism underlying the LHY and CCA1 regulated oil accumulation. Triacylglycerol (TAG) accumulation in Arabidopsis developing seeds was increased in LHY-overexpressing (LHY-OE) and decreased in lhycca1 plants compared to wild-type (WT). Metabolic tracking of lipids in developing seeds indicated that fatty acids (FAs) of major lipid precursors for TAG production increased more rapidly in LHY-OE and slowly in lhycca1 than in WT, suggesting that LHY enhanced FA synthesis. Transcript analysis revealed that the expression of genes involved in FA synthesis, including the one encoding {beta}-ketoacyl-ACP synthase III (KASIII), was oppositely changed in developing seeds of LHY/CCA1-OEs and those of lhycca1. Chromatin immunoprecipitation, electrophoretic mobility shift, and transactivation assays indicated that LHY directly bound and activated the promoter of KASIII. Furthermore, phosphatidic acid, a metabolic precursor to TAG, inhibited LHY binding to KASIII promoter elements. Our data reveal a new regulatory mechanism by the core clock regulators for storage lipid production during plant seed development.

Shikonin, a 1,4-naphthoquinone derivative produced by several Boraginaceae species, exhibits unique pharmacological properties and is used as a natural dye. The regulatory factors of shikonin production have been demonstrated using a cell culture system of Lithospermum erythrorhizon. Among these factors, copper is known to be the strongest enhancer of shikonin production. Although shikonin biosynthesis has been studied for over 40 years, the steps of naphthalene ring formation are still unknown, as is the reason for the effect of copper. In this study, we explored candidate genes associated with shikonin production using a PCR-select subtraction experiment. Polyphenol oxidase (PPO), a dicopper-dependent oxidoreductase, was highlighted because it showed synchronous expression with shikonin production. Transcriptome analysis of hairy roots and cultured cells of this plant revealed that, of the five PPO genes expressed in L. erythrorhizon, only PPO1 showed a strong correlation with shikonin production. Next, we generated genome-edited hairy roots of LePPO1 using CRISPR/Cas9-mediated mutagenesis to analyze its impact on shikonin derivative and other specialized metabolite production. The results showed that shikonin content was markedly reduced in all LePPO1-ge lines. Interestingly, the content of deoxyshikonofuran, a hydroquinone derivative and shunt product that branches after GHQ-3''-OH in the shikonin biosynthetic pathway, remained unaffected in the LePPO1-ge lines. These findings suggest that LePPO1 participates in naphthalene ring formation and explain why a copper ion is crucial for shikonin biosynthesis.

Physiological circadian rhythms are coordinated in the plant body in an orderly manner. The coordination of time information has been studied from the aspects of cell-cell local coupling and long-distance communication between tissues. These studies were based on the idea that the behavior of the clock gene circuit represents the physiological rhythms. Here we report the cellular circadian rhythm of a bioluminescence reporter which is not governed by the clock gene circuit in the expressing cells. Using a dual-color bioluminescence monitoring system in Lemna minor transfected with the AtCCA1::LUC+ and CaMV35S::PtRLUC reporters, cellular bioluminescence rhythms with different free-running periods (FRPs) were detected in the same cells. Co-transfection experiments with the two reporters and a clock gene overexpressing effector revealed that the circadian properties of the AtCCA1::LUC+ rhythm, but not those of the CaMV35S::PtRLUC rhythm, were altered in the cells with a dysfunctional clock gene circuit. This indicates that the AtCCA1::LUC+ rhythm is a direct output of the cellular circadian clock while the CaMV35S::PtRLUC rhythm is not. After plasmolysis, the CaMV35S::PtRLUC rhythm disappeared while the AtCCA1::LUC+ rhythm persisted. The plant circadian system consists of both cell-autonomous rhythms and non-cell-autonomous rhythms that are unaffected by the cellular clock.

In many flowering plants, the transition from vegetative growth to reproductive development is regulated by seasonal changes in photoperiod. Under inductive photoperiods, leaves produce the florigen FT (FLOWERING LOCUS T), which is transported to the shoot apex to promote flowering. The photoperiod is known to have a major effect on the flowering of chrysanthemum. In the perennial short-day (SD) plant Chrysanthemum seticuspe, the expression of CsFTL3 (FT-like gene) does not increase immediately after shifting from long-day (LD) to SD conditions but gradually accumulates under continuous SD conditions, peaking during inflorescence development. However, the underlying mechanism remains elusive. We show that CsFDL1 (an ortholog of FD) and CsFTL3 exhibit a significant inverse expression pattern in leaves during the initial stage of short-day inductions. Furthermore, the expression of CsFTL3 is upregulated in the leaves of CsFDL1-knockdown transgenic lines. CsFDL1 is expressed in leaves and forms a complex with CsFTL3 to recognize several TCGA- and ACGT-containing motifs in the CsFTL3 promoter. The CsFTL3-CsFDL1 complex downregulates CsFTL3 expression, thereby preventing its excessive induction by SD signals and inhibiting precocious floral transition. This study reveals that CsFDL1 acts as a key early repressor in the photoperiodic flowering pathway of chrysanthemum leaf, mediating negative feedback regulation by forming a complex with CsFTL3 to achieve precise temporal control of short-day-dependent flowering responses.

Rho/Rac of plant (ROP) GTPases are a plant-specific subfamily of Rho small GTP-binding proteins that function as molecular switches by being converted to the active state by guanine nucleotide exchange factors (GEFs) and to the inactive state by GTPase-activating proteins (GAPs). The bryophyte Marchantia polymorpha contains single-copy genes encoding ROP (MpROP), two types of GEFs (ROPGEF and SPIKE (SPK)), and two types of GAPs (ROPGAP and ROP enhancer (REN)). MpROP regulates the development of various organs, including the air chambers, rhizoids, and clonal propagule gemmae. While the sole PRONE-type ROPGEF, KARAPPO (MpKAR), plays an essential role in gemma initiation, little is known about the in-planta functions of other ROP regulatory factors in M. polymorpha. In this study, we focused on the functions of two types of GAPs: MpROPGAP and MpREN. Loss-of-function Mprenge single mutants showed pleiotropic defects in thallus growth, air chamber formation, rhizoid tip growth, and gemma development, whereas MpROPGAP mutants showed no detectable abnormalities. Despite the distinctive domain structures of MpROPGAP and MpREN, MpropgapgeMprenge double mutants showed more severe phenotypes than the Mprenge single mutants, suggesting redundant functions of MpROPGAP and MpREN in gametophyte organogenesis. Interestingly, overexpression of MpROPGAP, MpREN, and dominant-negative MpROP (MpROPDN) resulted in similar air chamber defects, as well as loss-of-function of MpREN and MpROPGAP and overexpression of constitutively active MpROP (MpROPCA), suggesting importance of activation/inactivation cycling (or balancing) of MpROP. Furthermore, we proved the contributions of the sole DOCK family GEF, MpSPK, to MpROP-regulated air chamber formation. In summary, our results demonstrate a significant role of the two GAPs in the development of various organs and that the two GEFs are responsible for organogenesis through the control of the MpROP active/inactive cycle in the vegetative growth of M. polymorpha.

O_LIMomilactones and phytocassanes are diterpenoid phytoalexins involved in plant chemical defense. These metabolites, along with biosynthetic gene clusters (BGCs), are conserved in wild rice. However, the mechanisms by which phytoalexins are regulated in wild rice are unclear. Thus, we aimed to investigate the regulatory mechanisms for biosynthetic genes within the BGCs of diterpenoid phytoalexins. C_LIO_LIWe conducted a transcriptome analysis of five wild rice species, Oryza rufipogon, Oryza punctata, Oryza officinalis, Oryza brachyantha, and Leersia perrieri, after CuCl2 treatment. C_LIO_LIAmong the CuCl2-responsive transcription factors, diterpenoid phytoalexin factor (DPF), which regulates phytoalexin production in cultivated rice (Oryza sativa), was broadly conserved in wild rice and showed phytoalexin-inducing activity when introduced into cultivated rice. Highly conserved genomic regions containing N-boxes (5'-CACGAG-3'), the potential binding motif of DPF, were found. CRISPR/Cas9 genome editing to remove these regions showed that biosynthetic gene expression and phytoalexin production were significantly attenuated after CuCl2 treatment in the leaves of the edited plants. Thus, the cis-trans factor combination of DPF and N-boxes is a key determinant of regulation. C_LIO_LIDPF has evolved as a strong cis-trans regulatory system for diterpenoid phytoalexin production, with N-boxes generated within the cluster region during the evolution from wild rice to cultivated rice. C_LI

Dark-germinated angiosperms develop the chloroplast precursors called etioplasts in cotyledon cells. Etioplasts develop lattice membrane structures called prolamellar bodies (PLBs), where the chlorophyll intermediate protochlorophyllide (Pchlide) forms a ternary complex with NADPH and light-dependent NADPH-Pchlide oxidoreductase (LPOR). The lipid bilayers of etioplast membranes are mainly composed of galactolipids, which play important roles in membrane-associated processes in etioplasts. Although etioplast membranes also contain two anionic lipids, phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQDG), the roles of these anionic lipids are unknown. To reveal the importance of PG and SQDG for the development of etioplasts, we characterized etiolated Arabidopsis mutants deficient in the biosynthesis of PG and SQDG. A partial deficiency in PG biosynthesis loosened the lattice structure of PLBs and impaired the insertion of Mg2+ into protoporphyrin IX, leading to a significant decrease in Pchlide content. Although a complete lack of SQDG biosynthesis did not notably affect both PLB formation and Pchlide biosynthesis, the lack of SQDG in addition to the partial deficiency of PG caused strong impairments of these processes. The results suggested that PG is required for PLB formation and Pchlide biosynthesis, whereas SQDG plays an auxiliary role in these processes. Notably, the PG deficiency and the lack of SQDG oppositely affected the dynamics of LPOR complexes after photoconversion, suggesting different involvements of PG and SQDG in the organization of LPOR complexes. Our data demonstrate pleiotropic roles of anionic lipids in etioplast development.

NIMA-related kinases (NEKs) regulate a series of mitotic events in fungi and animals, whereas plant NEKs regulate growth direction of cells and organs. The liverwort Marchantia polymorpha has a single functional MpNEK1 gene, whose knockout leads to twisted growth of rhizoids. MpNEK1 is also expressed in the meristem of vegetative flat organ, thallus, while its function remains unknown. Here, we generated transgenic lines for the inducible expression of MpNEK1 using an estrogen receptor mediated system. Estradiol treatment efficiently induced the accumulation of MpNEK1 mRNA and MpNEK1-Citrine fusion protein throughout plant body. Overexpression of MpNEK1 severely suppressed growth of rhizoids and thalli, eventually causing the lethality of juvenile plants. The effect of estradiol was reversible until 3 days, whereas 7-days treatment resulted in irreversible suppression of growth. This severe effect was observed even at the nanomolar level of estradiol. EdU staining and microtubule imaging clearly indicated the suppression of cell proliferation by estradiol-induced MpNEK1. Unexpectedly, the overexpression of kinase-deficient MpNEK1 also suppressed thallus growth and rhizoid formation, despite their slightly mild effect than the full length MpNEK1, indicating phosphorylation-independent mechanism of growth suppression. In conclusion, overexpression of MpNEK1 suppresses cell division and elongation, leading to growth cessation and lethality. Our results imply that the expression of MpNEK1 is tightly regulated and plant NEKs might control cell division as in fungi and animals.

Bioactive specialized metabolites (SMs) are synthesized and sequestered in specific cellular compartments or organelles as a self-defense strategy against their intrinsic toxicity. Liverwort-specific oil bodies accumulate large amounts of SMs and contribute to chemical defense; however, the molecular mechanisms underlying SM sequestration in oil bodies remain largely unknown. Therefore, in this study, we focused on MpABCG1 and MpABCG36, which are ATP-binding cassette (ABC) protein family members localized to the oil bodies of liverwort Marchantia polymorpha. Sesquiterpene (thujopsene, chamigrene, and himachalane) accumulation was reduced in the Mpabcg1 and Mpabcg36 loss-of-function mutants. Notably, levels of the bisbibenzyls, marchantins C and A, were predominantly reduced in Mpabcg1, but not in Mpabcg36. Although the Mpabcg1 mutant formed a number of oil bodies labeled with mCitrine- MpSYP12B (oil body membrane marker) comparable to that of the wild-type, the number of oil bodies stained with BODIPY 493/503, which has an affinity for lipophilic SMs, was reduced. This finding suggests that MpABCG1 and MpABCG36 mutations affect SM accumulation in the oil body but have little impact on oil body formation. Overall, our results highlight the involvement of MpABCG1 and MpABCG36 in the accumulation of SMs and/or their precursors in liverwort oil bodies.

Dark-induced senescence triggers significant metabolic changes that recycle resources and ensure plant survival. In this study, we identified a transcription factor OsS40-14 in rice, which can form homo-oligomers. The oss40-14 knockout mutants exhibited stay-green phenotype of primary leaf and flag leaf during dark-induced condition, with substantial retention of chlorophylls and photosynthetic capacity as well as remarkably reduced reactive oxygen species (ROS), while OsS40-14 overexpressing transgenic lines (oeOsS40-14) showed an accelerated senescence phenotype under dark-induced leaf senescence conditions. Transcriptome analysis revealed that when the detached leaves of oss40-14 and WT were treated in darkness condition for 72 hours, 1585 DEGs (|Log2FC| [&ge;]1, P value<0.05) were reprogrammed in oss40-14 relative to WT. CUT&Tag-seq analysis in protoplast transient expression of OsS40-14 system showed that OsS40-14 was 40.95% enriched in the transcription start site (TSS) of the genome. Sequence clustering analysis showed that OsS40-14 protein was mainly enriched and bound to TACCCACAAGACAC conserved elements. The seed region "ACCCA" of OsS40 proteins was identified by single nucleotide mutagenesis EMSA. The integrative analysis of transcriptome and CUT&Tag-seq datasets showed 153 OsS40-14-targeted DEGs, they mainly enriched in plastid organization and photosynthesis process at dark-induced condition in oss40-14 relative to WT. Among them, eleven candidate targets of OsS40-14 such as Glucose 6-phosphate/phosphate translocator, Na+/H+ antiporter, Catalase, Chitinase 2, Phosphate transporter 19, OsWAK32, and OsRLCK319 were directly targeted and upregulated confirmed by ChIP-PCR and RT-qPCR. It demonstrates a novel model of OsS40-14 mediating macromolecule metabolism and nutrient recycling controls the plastid organization during dark-induced leaf senescence. Significant statementInvolvement of OsS40-14 in macromolecule catabolism, nutrient recycling, and ROS homeostasis revealed a plastid organization defection of dark-induced senescence in rice

Hevea brasiliensis and Taraxacum kok-saghyz are sources of nature rubber (NR) and recent research has focused on finding a method to increase NR quality and production. NR consists mainly of cis-1,4-polyisoprene, which is synthesized from sucrose by a series of reactions during carbon metabolism. WRINKLED1 (WRI1) is a transcription factor (TF) that coordinates many genes involved in carbon metabolism and lipid biosynthesis. Here, we isolated and characterized two orthologues of WRI in rubber-producing plants H. brasiliensisa and T. kok-saghyz, which are highly expressed in their latex. Subcellular localization and ectopic expression in Arabidopsis thaliana indicated that the TFs, HbWRI1 and TkWRI1, are involved in lipid accumulation. Overexpression of HbWRI1 and TkWRI1 in T. kok-saghyz substantially enhanced NR quality and production, including dry rubber content, molecular weight, and the diameter of rubber particles in the latex. Conversely, they were decreased in TkWRI1 repression. Furthermore, based on the activated expression in transgenic T. kok-saghyz latex and the existence of AW-box element in the promoter regions, 16 direct downstream genes of HbWRI1 and TkWRI1 were identified by a dual-luciferase reporter assay and yeast one-hybrid assay, and their products were responsible for both NR biosynthesis and lipid metabolism. These results reveal a regulatory module that HbWRI1 and TkWRI1 TFs can positively regulate NR quality and production in T. kok-saghyz latex by synergistically regulating the entire NR biosynthesis. This module is expected to be utilized to develop superior varieties to enhance NR quality and production.

In C4 plants, mesophyll (M) chloroplasts aggregate toward bundle sheath (BS) cells in response to environmental stress, which would contribute to C4 photosynthetic cycle adjustment between M and BS cells. However, it remains unclear whether mesophyll chloroplast movement is an intercellular response mediated by BS cells. One major challenge to resolving this is the difficulty in observing how M chloroplasts aggregate toward adjacent BS cells due to scattering and absorption of observation light in live-leaf tissues. We established a live leaf-section imaging technique that enables the long-term observation of sections of chemically unfixed leaf blades, with which we quantitatively analyzed M chloroplast movements. Another challenge in clarifying the contribution of BS cells to M chloroplast movement is the selective ablation of BS cells without impairing their function of M cells. To investigate the necessity of BS cells for M chloroplast movement, we developed a method to remove BS cells only based on differences in shape and size between M and BS cells. We also found that chloroplasts in M cells without adjacent BS cell contents did not show typical aggregative movement but showed a light-avoidance response. This indicates that the M chloroplast aggregative movement occurs during communication with BS cells. HighlightWe established live leaf-section imaging to observe individual chloroplast movements in multi-layered cells and found that bundle sheath cells are involved in the aggregative movement of mesophyll chloroplasts.

Plants continuously develop shoot branches derived from axillary meristems. In rice (Oryza sativa), TILLERS ABSENT1 (TAB1), an ortholog of Arabidopsis WUSCHEL, plays an essential role in axillary meristem formation by promoting stem cell proliferation. Although several genes associated with TAB1 function have been identified, the molecular mechanisms underlying stem cell proliferation during axillary meristem formation remain poorly understood. Here we identify ABERRANT SPIKELET AND PANICLE1 (ASP1), a TOPLESS-like transcriptional corepressor, as a novel regulator of axillary meristem formation, and investigate downstream mechanisms regulated by TAB1 and ASP1. In asp1, the stem cell region was expanded, indicating that ASP1 negatively regulates stem cell proliferation. Notably, WOX4, a paralog of TAB1, was precociously expressed in asp1, possibly in association with expansion of the stem cell region. Genetic analysis further revealed that asp1 mutation rescued the loss of axillary meristems in tab1. Transcriptome analysis showed that several type-A RESPONSE REGULATOR (OsRR) genes, encoding negative regulators of cytokinin signaling, were upregulated in tab1 relative to wild type, asp1, and the tab1 asp1 double mutant. Consistently, fluorescence of the synthetic cytokinin reporter was absent during axillary meristem formation in tab1 but was detected in wild type and tab1 asp1. Moreover, overexpression of OsRR10 inhibited axillary meristem formation, phenocopying tab1. Collectively, these findings suggest that TAB1 activates cytokinin signaling by repressing type-A OsRR expression, whereas ASP1 negatively regulates cytokinin signaling by promoting the expression of these genes. Thus, rescue of the tab1 phenotype by asp1 mutation probably reflects restoration of cytokinin signaling.

Heterotrimeric GTP-binding proteins (G proteins) are a group of regulators essential for signal transmission into cells. AtRGS1 (Regulator of G protein Signaling 1) with intrinsic GTPase-accelerating protein (GAP) activity could suppress G protein and glucose signal transduction in Arabidopsis. However, how AtRGS1 activity is regulated is currently poor understood. Here we identified a knockout mutant orp2a-1 (oxysterol-binding protein (OSBP)-related protein 2A) which shows phenotypes similar to agb1-2 (arabidopsis g-protein beta 1). With overexpression of ORP2A, transgenic lines display short hypocotyl, hypersensitivity to sugar and lower intracellular AtRGS1 level than control. Consistently, ORP2A shows interaction with AtRGS1 in vitro and vivo. Tissue specificity of ORP2A with two alternative protein forms imply its functions in organ size and shape controlling. Bioinformatic data and phenotypes of orp2a-1, agb1-2 and double mutant reveal genetic interactions in the regulation of G protein signaling and sugar response between ORP2A and G{beta}. Both alternative splicing forms of ORP2A locate in the ER, PM (Plasma Membrane) and EPCS (ER-PM Contact Sites), and interact with VAP27-1 mediated by a FFAT-like motif in vivo and vitro. ORP2A also displays differential phosphatidyl phosphoinositide binding activity mediated by its PH domain in vitro. Taken together, it is suggested that Arabidopsis membrane protein ORP2A interacts with AtRGS1 and VAP27-1 to positively regulate G protein and sugar signaling by facilitating AtRGS1 degradation.

Recent global marine lipidomic analysis reveals a strong relationship in the ocean between temperature and phytoplanktonic abundance of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are essential for human nutrition and primarily from phytoplankton in marine food webs. In phytoplanktonic organisms, EPA may play a major role in regulating the phase transition temperature of membranes, while the function of DHA remains to be explored. In the oleaginous diatom Phaeodactylum tricornutum, DHA is distributed mainly on extraplastidial phospholipids, which is very different from the EPA enriched in thylakoid lipids. Here, CRISPR/Cas9-mediated knockout of ptELO5a, which encodes a delta-5 elongase catalyzing the elongation of EPA to synthesize DHA, led to a substantial interruption of DHA synthesis in P. tricornutum. The ptELO5a mutants show significant alterations in transcriptome and glycerolipidomes including membrane lipids and triacylglycerols under normal temperature (22{degrees}C), and are more sensitive to elevated temperature (28{degrees}C) than wild type. We conclude that the PtELO5a-mediated synthesis of small amounts of DHA has indispensable functions in regulating the membrane lipid, and indirectly contributing storage lipid accumulation and maintaining thermomorphogenesis in P. tricornutum. This study also highlights the significance of DHA synthesis and lipid composition for environmental adaptation of P. tricornutum.

Phosphoinositides (PIs) as regulatory membrane lipids play essential roles in multiple cellular processes. Although the exact molecular targets of PIs-dependent modulation remain largely elusive, the effects of disturbed PIs metabolism could be employed to propose regulatory modules associated with particular downstream targets of PIs. Here, we identified the role of GRAIN NUMBER AND PLANT HEIGHT 1 (GH1), which encodes a suppressor of actin (SAC) domain-containing phosphatase with unknown function in rice. Endoplasmic reticulum-localized GH1 specifically dephosphorylated and hydrolyzed phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Inactivation of GH1 resulted in massive accumulation of both PI4P and PI(4,5)P2, while excessive GH1 caused their depletion. Notably, superabundant PI4P and PI(4,5)P2 could both disrupt actin cytoskeleton organization and suppress cell elongation. Interestingly, both PI4P and PI(4,5)P2 inhibited actin-related proteins 2 and 3 (Arp2/3) complex-nucleated actin branching networks in vitro, whereas PI(4,5)P2 showed more dramatic effect in a dose-dependent manner. Overall, the overaccumulation of PI(4,5)P2 resulted from dysfunction of SAC phosphatase possibly perturbs Arp2/3 complex-mediated actin polymerization, thereby disordering the cell development. These findings imply that Arp2/3 complex might be the potential molecular target of PI(4,5)P2-dependent modulation in eukaryotes, thereby providing new insights into the relationship between PIs homeostasis and plants growth and development.

Thermospermine, a structural isomer of spermine, suppresses auxin-inducible xylem differentiation, whereas spermine is implicated in stress responses in angiosperms. Thermospermine synthase ACAULIS5 (ACL5) is well conserved from algae to land plants, but its physiological function remains elusive in non-vascular plants. Here we focused on MpACL5, a gene in the liverwort Marchantia polymorpha, which rescued the dwarf phenotype of the acl5 mutant of Arabidopsis. In the Mpacl5 mutants generated by genome editing, growth of the vegetative organ, thallus, and the sexual reproductive organ, gametangiophore, was severely retarded. The mutant gametangiophore exhibited remarkable morphological defects such as short stalks, fasciation, and indeterminate growth; it was formed as a fusion of two gametangiophores and a new gametangiophore was often initiated from the old one. Furthermore, Mpacl5 was shown to be hypersensitive to heat and salt stresses. Given the absence of spermine in liverworts including M. polymorpha, these results reveal that thermospermine has a dual primordial function in organ development and stress responses in the basal land plant lineage, the latter of which may have eventually been assigned to spermine during the land plant evolution.

Much efforts have been devoted to identify options to improve plant photosynthetic capacity. Most of these engineering focuses more on enhancing photosynthetic capacity of the mesophyll cells. In this study, we specifically expressed CYTOKININRESPONSIVE GATA FACTOR 1 (CGA1) in the vascular bundle and bundle sheath cells (ProGLDPA: CGA1) which generally have low photosynthetic capacity. The introduction of CGA1 resulted in increased numbers and sizes of chloroplasts in both bundle sheath and vascular bundle, without noticeable difference in these properties in mesophylls. Additionally, compared with wild type (WT), transgenic lines showed enhanced development of mitochondria and peroxisomes. Leaf photosynthetic rates in these transgenic lines were higher than those in WT, especially under high light. Furthermore, the amount of RuBisCO, Vcmax, Jmax, and the number of starch granules were also significantly increased in the transgenic lines, which led to about 20% increase in biomass. Remarkably, the CO2 compensation point of ProGLDPA: CGA1 decreased compared with WT, resembling the characteristics of proto-Kranz during C4 evolution. All these results indicated that enhancing photosynthetic properties of vascular bundle and bundle sheath cell is an effective approach to improve leaf photosynthesis, and highlighted that utilization of the cell lacking photosynthetic capacity is a promising way to improve plant photosynthesis for greater capacity.